Specific mosaic KRAS mutations affecting codon 146 cause oculoectodermal syndrome and encephalocraniocutaneous lipomatosis.

Oculoectodermal syndrome (OES) and encephalocraniocutaneous lipomatosis (ECCL) are rare disorders that share many common features, such as epibulbar dermoids, aplasia cutis congenita, pigmentary changes following Blaschko lines, bony tumor-like lesions, and others. About 20 cases with OES and more than 50 patients with ECCL have been reported. Both diseases were proposed to represent mosaic disorders, but only very recently whole-genome sequencing has led to the identification of somatic KRAS mutations, p.Leu19Phe and p.Gly13Asp, in affected tissue from two individuals with OES. Here we report the results of molecular genetic studies in three patients with OES and one with ECCL. In all four cases, Sanger sequencing of the KRAS gene in DNA from lesional tissue detected mutations affecting codon 146 (p.Ala146Val, p.Ala146Thr) at variable levels of mosaicism. Our findings thus corroborate the evidence of OES being a mosaic RASopathy and confirm the common etiology of OES and ECCL. KRAS codon 146 mutations, as well as the previously reported OES-associated alterations, are known oncogenic KRAS mutations with distinct functional consequences. Considering the phenotype and genotype spectrum of mosaic RASopathies, these findings suggest that the wide phenotypic variability does not only depend on the tissue distribution but also on the specific genotype.

[Homozygous and compound heterozygous RYR1 mutations. New findings on prevalence and penetrance of malignant hyperthermia]. / Homozygote und "compound"-heterozygote RYR1-Mutationen. Neue Erkenntnisse zu Prävalenz und Penetranz der malignen Hyperthermie.

BACKGROUND: Malignant hyperthermia (MH) is a life-threatening, acute pharmacogenetic disorder mostly due to heterozygous mutations in the ryanodin receptor 1 (RYR1) gene. Diagnosis is generally confirmed by the in vitro contracture test (IVCT). In this study the genotype-phenotype correlation was analyzed and the presumed prevalence of MH is discussed. PATIENTS AND METHODS: After the diagnosis of MH susceptibility by the IVCT DNA samples of 44 patients were analyzed for mutations in the RYR1 gene using the polymerase chain reaction and sequencing. For genotype-phenotype correlation, the mutation analysis data were compared with the IVCT data. RESULTS: Out of the 44 patients tested 13 were identified with a heterozygous mutation, 1 patient with a homozygous mutation (c.1840C>T) and 1 patient with compound heterozygous mutations (c.1840C>T and c.6487C>T). The two patients with two mutated alleles showed a stronger response in the IVCT compared to those with only one mutated allele. Patients with one RYR1 mutation displayed significantly higher contractures in the IVCT than patients without RYR1 mutations. CONCLUSION: In the two patients described the presence of two mutated RYR1 alleles seemed to have an additive effect on the functional restriction of the (RYR1 receptor and to lead to a stronger response both in the IVCT and with regard to clinical signs. The patients with no detected RYR1 mutations possibly have a RYR1 mutation with smaller effects outside the hot spot regions tested and/or false positive IVCT results. The data from a small patient group indicate a substantially higher prevalence of MH with a correspondingly lower penetrance in the German population than previously assumed.

A boy with classical Rubinstein-Taybi syndrome but no detectable mutation in the CREBBP and EP300 genes.

Rubinstein-Taybi syndrome (RTS) is a rare autosomal dominant genetic disorder and is characterized by mental retardation, distinctive facial features, broad and often angulated thumbs and great toes. We report on a 7 year old boy with classical Rubinstein-Taybi syndrome. His facial and clinical features were very typical, including broad thumbs with radial angulation and broad great toes. Rigorous genetic analysis of the CREBBP and EP300 genes using DNA sequencing and multiple ligation-dependent probe amplification (MLPA) revealed no causative mutation in this boy, only a hitherto unreported but paternally inherited heterozygous sequence alteration, c.506 1+9C>T in IVS 30-31, which most likely represents a normal variant (NetGene 2 splice prediction software). We question if this boy could have a hitherto undetectable mutation type.

Prenatal diagnosis of a recombinant chromosome 7 resulting in trisomy 7q11.22 --> qter.

Prenatal diagnosis of trisomy 7 is complex due to only a few reported cases. We report here on a stillborn boy with very large duplication of 7q11.22 --> qter, encompassing almost the entire long arm of chromosome 7. Ultrasound, fetal and parental chromosome banding, fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (CGH) analyses were performed. Sonographic findings included growth retardation, micrognathia, ventricular septal defect (VSD), aortic coarctation, bradyarrhythmia, pericardial effusion, bilateral hydronephrosis, infravesical obstruction, and cerebellar hypoplasia. Chromosome analysis after cordocentesis at 23 weeks of gestation revealed an abnormal male karyotype with 46 chromosomes and a derivative chromosome 7 with a very large duplication of the long arm, 46,XY,der(7)(qter --> q11.2::p22 --> qter). The mother was found to carry an apparently balanced pericentric inversion, 46,XX,inv(7)(p22q11.2). Thus, the recombinant chromosome 7 [rec(7)dup(7q)inv(7)(p22.3q11.22)mat] of the fetus must have arisen through meiotic crossing-over between the inverted chromosome and the normal chromosome 7 in the maternal germline. FISH and array CGH results confirmed the recombinant chromosome 7 in the fetus and indicated a loss of 1.9 Mb at chromosome 7pter --> p22.3 (pter to 1,948,072 bp), and a gain of 87.04 Mb at chromosome 7q11.22 --> qter (71,760,154 bp to qter). The rare syndrome of almost complete trisomy 7q may be suspected in cases of growth retardation, cerebellar hypoplasia, micrognathia, aortic coarctation and VSD and hydronephrosis. Invasive prenatal diagnosis must be offered to the parents.

GJB2 mutations and genotype-phenotype correlation in 335 patients from germany with nonsyndromic sensorineural hearing loss: evidence for additional recessive mutations not detected by current methods.

We report on 335 patients (319 families) with mild-to-profound nonsyndromic sensorineural hearing loss. We identified 178 mutated GJB2 alleles representing 29 different sequence changes (including 3 novel mutations: Q7P, N14D, H100Q), and 2 alleles with the deletion del(GJB6-D13S1830) of the GJB6 gene. Eleven GJB2 mutations (119 mutated alleles) were truncating (T), and 18 mutations (59 alleles) were nontruncating (NT). Biallelic GJB2 mutations were found in 71 patients (21.2%; 67 families; 25 different genotypes). Audiograms of 62 patients (56 families) with biallelic GJB2 mutations typically indicated a profound hearing loss with T/T mutations, moderate hearing loss with T/NT mutations, and mild hearing impairment with NT/NT mutations (p < 0.01, Student's t test). From 37 patients (34 families) with biallelic GJB2 mutations, audiograms at different ages were available and indicated progressive hearing loss (>15 dB) in 10 patients (27.0%, 10 families). Interestingly, we identified an unexpectedly large subset of patients (n = 29; 8.7%) presenting with only one GJB2 mutation (n = 14 T/wild-type; n = 15 NT/wild-type). This strongly suggests the presence of additional recessive mutations that are not detected by current GJB2 mutation and GJB6 deletion analyses.

Haploinsufficiency of 16.4 Mb from chromosome 22pter-q11.21 in a girl with unilateral conductive hearing loss.

We present the postnatal diagnosis of a de novo der(18)t(18;22)(p11.32;q11.21)pat, resulting in an unbalanced 45,XX,der (18)t(18;22) karyotype in a girl with conductive hearing loss on the left and ptosis of the right upper eye-lid. Unilateral ptosis was also observed in the patient's 2 years and 8 months younger sister, who grows noticeably faster and appears to be a much quicker learner. After speech therapy the patient was eventually placed in normal school. The haploinsufficient 16.4-Mb region on chromosome 22pter-->q11.21 contains 10 genes as well as many predicted genes, pseudogenes, and retrotransposed sequences with unknown functions. This observation may prove useful for prenatal diagnosis and genetic counselling of chromosome 22q11.1 gains and losses.

Epimutation at human chromosome 14q32.2 in a boy with a upd(14)mat-like clinical phenotype.

Recently, three reports described deletions and epimutations affecting the imprinted region at chromosome 14q32.2 in individuals with a phenotype typical for maternal uniparental disomy of chromosome 14 [upd(14)mat]. In this study, we describe another patient with upd(14)mat-like phenotype including low birth weight, neonatal feeding problems, muscular hypotonia, motor and developmental delay, small hands and feet, and truncal obesity. Conventional cytogenetic analyses, fluorescence in situ hybridization subtelomere screening, multiplex ligation-dependent probe amplification analysis of common microdeletion and microduplication syndromes, and methylation analysis of SNRPN all gave normal results. Methylation analysis at 14q32.2 revealed a gross hypomethylation of the differentially methylated regions (intergenic DMR and MEG3-DMR). Further molecular studies excluded full or segmental upd(14)mat as well as a microdeletion within this region. Evidently, the upd(14)mat-like clinical phenotype is caused by an epimutation at 14q32.2. The clinical and molecular features of this novel case are discussed with respect to the recently published cases.

A severely mentally and motor retarded girl with monosomy 3pter-->p25 and trisomy 8q24-->qter due to a familial reciprocal translocation t(3;8)(p25;q24).

A severely mentally and motor retarded girl with monosomy 3pter--p25 and trisomy 8q24-qter due to a familial reciprocal translocation t(3;8) (p25;q24): We report a familial translocation t(3;8) in a three generation family that includes a severely retarded 9-year-old girl with intrauterine and postnatal growth retardation, microcephaly, capillary hemangiomas of the forehead and perioral region, synophrys, ptosis, long philtrum, high arched palate, micrognathia, malformed ears, clinodactyly, hypotonia, mental and motor retardation. The pedigree was highly suggestive ofa familial rearrangement. Cytogenetics and fluorescent in situ hybridization (FISH) showed an unbalanced translocation of chromosomes 3p25 and 8q24 of maternal origin, karyotype 46,XX,der(3)t(3;8)(p25q24)mat. Using FISH the breakpoint at 8q24 was located distal of TRPS1, the gene for trichorhinophalangeal syndrome. The balanced translocation was found in the mother, maternal grandmother and prenatally diagnosed brother. Ten individuals (seven miscarriages, niece, two nephews) probably also had an unbalanced translocation. Genetic counseling was given to the family. Because of the hemizygous deletion of the VHL gene at chromosome 3p25.3, the patient is at risk for von Hippel-Lindau (VHL) syndrome, predisposing to retinal, cerebellar, spinal haemangioblastomas, renal cell carcinoma, phaeochromocytoma and pancreatic tumors. Therefore, for early detection and treatment of VHL syndrome, we performed periodic screening beginning at age 5 years. A familial translocation t(3;8) is very rare and there are no previous reports on terminal monosomy 3p (pter-->p25) and terminal trisomy 8q (q24-->qter).

Rubinstein-Taybi syndrome and CREBBP c.201 202delTA mutation: a case presenting with varicella meningoencephalitis.

Rubinstein-Taybi syndrome (RTS) is a rare syndrome with a frequency of approximately 1 in 125,000 affected newborns, which is characterized by mental retardation, growth retardation, a particular dysmorphology and, in a subset of cases, immunodeficiency. RTS is typically caused by CREBBP deficiency, and heterozygous mutation or deletion of the CREBBP gene has been identified in 60-70% of patients. The inheritance is autosomal dominant but reports of vertical transmission are exceedingly rare; near-all cases are caused by de novo mutations. Here we present an 8-month-old boy with varicella meningoencephalitis, RTS, and a de novo deletion of the CREBBP gene of two base pairs at position 201-202 in exon 2, c. 201 202delT. The mutation has not been described previously but it predicts a protein truncation, and truncating CREBBP mutations are typical causes of RTS.

Boy with seizures (West syndrome) and distal 7q duplication syndrome due to an unbalanced 7q;9p translocation.

We report a 15 month old boy with prominent metopic suture, epicanthal folds, strabismus, low-set ears, microretrognathia, large anterior fontanel, bilateral simian creases, muscular hypotonia, and severe psychomotor retardation. He also had West syndrome. An electroencephalogram showed hypsarrythmia, and cranial MR indicated a myelinisation delay. Standard karyotyping showed additional material on one chromosome 9p. Using FISH, a terminal 7q duplication spanning 26 Mb in size and a terminal 9p deletion sized (at least) 9.1 Mb were identified. The father had a karyotype of t(7;9)(q33;p23) and the mother's karyotype was normal. The boy presented typical facial features of the distal 7q duplication syndrome but no genital anomalies attributable to his distal 9p deletion. We assume that the severe epilepsy is likely due to the trisomy 7q.

Phosphorylation and activation of B-Myb by cyclin A-Cdk2.

BACKGROUND: Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle. Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified. B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells. Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle. In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks. RESULTS: We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein. Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region. Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase. CONCLUSIONS: This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery. We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb. Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.

Two independent chromosomal rearrangements, a very small (550 kb) duplication of the 7q subtelomeric region and an atypical 17q11.2 (NF1) microdeletion, in a girl with neurofibromatosis.

Most patients with neurofibromatosis (NF1) are endowed with heterozygous mutations in the NF1 gene. Approximately 5% show an interstitial deletion of chromosome 17q11.2 (including NF1) and in most cases also a more severe phenotype. Here we report on a 7-year-old girl with classical NF1 signs, and in addition mild overgrowth (97th percentile), relatively low OFC (10th-25th percentile), facial dysmorphy, hoarse voice, and developmental delay. FISH analysis revealed a 17q11.2 microdeletion as well as an unbalanced 7p;13q translocation leading to trisomy of the 7q36.3 subtelomeric region. The patient's mother and grandmother who were phenotypically normal carried the same unbalanced translocation. The 17q11.2 microdeletion had arisen de novo. Array comparative genomic hybridization (CGH) demonstrated gain of a 550-kb segment from 7qter and loss of 2.5 Mb from 17q11.2 (an atypical NF1 microdeletion). We conclude that the patient's phenotype is caused by the atypical NF1 deletion, whereas 7q36.3 trisomy represents a subtelomeric copy number variation without phenotypic consequences. To our knowledge this is the first report that a duplication of the subtelomeric region of chromosome 7q containing functional genes (FAM62B, WDR60, and VIPR2) can be tolerated without phenotypic consequences. The 17q11.2 microdeletion (containing nine more genes than the common NF1 microdeletions) and the 7qter duplication were not accompanied by unexpected clinical features. Most likely the 7qter trisomy and the 17q11.2 microdeletion coincide by chance in our patient.

Two complementary recombinant chromosomes 5 in a healthy woman.

We report a healthy woman with two abortions who is a carrier for a rare heterozygous double recombinant of an inv(5) chromosome, karyotype 46,XX,rec(5)dup(5p) inv(5)(p13q22),rec(5)dup(5q)inv(5)(p13q22). Her father had a 46,XY,inv(5)(p13q22) karyotype; his consanguineous wife had died. Molecular investigation of 11 highly polymorphic markers spanning chromosome 5 revealed biparental inheritance for two markers (D5S406, D5S681) on 5p15.3 and 5q13.1, and an allele constellation not compatible with paternal heterodisomy for marker D5S623 on 5q11.2. Eight markers were not informative. Three mechanisms of formation are proposed: First, fertilization of a normal oocyte by a sperm carrying the two recombinant chromosomes 5, followed by postzygotic recombination between the normal maternal homologue and the rec(5)dup(5p), and by loss of the mitotically recombined maternal homologue, leading to segmental paternal heterodisomy 5q13-->qter (trisomic rescue). Second, postzygotic recombination in a 46,XX,inv(5)(p13q22) zygote resulting in the 46,XX,rec(5)dup(5p)inv(5)(p13q22),rec(5) dup(5q)inv(5)(p13q22) karyotype, followed by absence of the original cell line in lymphocytes. Third and most likely, both parents were inv(5) carriers and complementary recombinations in maternal and paternal meiosis resulted in a zygote with two recombinant chromosomes 5. Our patient refused any further studies but later reported the birth of a phenotypically normal child. This is the first report known to us of complementation by two non-homologous recombinant chromosomes in a phenotypically normal woman, and the first example of a child born to a carrier of complementary recombinant chromosomes.

FISH studies in 45 patients with Rubinstein-Taybi syndrome: deletions associated with polysplenia, hypoplastic left heart and death in infancy.

Rubinstein-Taybi syndrome (RTS) is a dominant Mendelian disorder characterised by mental retardation, a typical facies, broad thumbs and short stature. Previous reports indicated that 4-25% of RTS patients have a submicroscopic 16p13.3 deletion of the CBP gene. Using FISH and cosmid probes RT100, RT191 and RT203 we studied 45 RTS patients from Germany, the Czech Republic, Austria and Turkey and found four deletions (8.9%, pooled data including other studies: 11%). All deletions were interstitial; three spanned the CBP gene (RT100-RT203) and one was smaller (RT100 only). Previous studies reported no phenotype-genotype correlation between RTS patients with or without a deletion. Our findings suggest a more severe phenotype. The mean age at presentation was 0.96 years in patients with a deletion as against 11.12 years in those without. Patients A and B with a deletion died in infancy which is rare in RTS and was not observed among the other patients. Patients A and D had accessory spleens, Patient A with hypoplastic left heart, abnormal pulmonary lobulation and renal agenesis. This is the second report of hypoplastic left heart and the first report of polysplenia with RTS. The signs suggest a developmental field defect (disturbance of laterality) either as a newly recognised pattern of RTS, or alternatively a novel contiguous gene syndrome.

Microphthalmia with linear skin defects syndrome (MLS): a male with a mosaic paracentric inversion of Xp.

The microphthalmia with linear skin defects syndrome (MLS) is an X-linked dominant disorder with male lethality. In the majority of the patients reported, the MLS syndrome is caused by segmental monosomy of the Xp22.3 region. To date, five male patients with MLS and 46,XX karyotype ("XX males") have been described. Here we report on the first male case with MLS and an XY complement. The patient showed agenesis of the corpus callosum, histiocytoid cardiomyopathy, and lactic acidosis but no microphthalmia, and carried a mosaic subtle inversion of the short arm of the X chromosome in 15% of his peripheral blood lymphocytes, 46,Y,inv(X)(p22.13 approximately 22.2p22.32 approximately 22.33)[49]/46,XY[271]. By fluorescence IN SITU hybridization (FISH), we showed that YAC 225H10 spans the breakpoint in Xp22.3. End-sequencing and database analysis revealed a YAC insert of at least 416 kb containing the genes HCCS and AMELX, and exons 2-16 of ARHGAP6. Molecular cytogenetic data suggest that the Xp22.3 inversion breakpoint is located in intron 1 of ARHGAP6, the gene encoding the Rho GTPase activating protein 6. Future molecular studies in karyotypically normal female MLS patients to detect submicroscopic rearrangements including the ARHGAP6 gene as well as mutation screening of ARHGAP6 in patients with no obvious chromosomal rearrangements will clarify the role of this gene in MLS syndrome.

Familial Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) occurs sporadically; however, at least four familial cases of WBS have been described previously. We describe a mother and her son with typical WBS. The diagnosis of WBS in the son was confirmed by molecular cytogenetic analysis fluorescence in situ hybridization. He had a deletion of 7q11.23 at the ELN locus. The mother was diagnosed after the identification of WBS in her affected son. She is deceased and was thus not studied by FISH. However, her combined symptoms make it very clear that she had WBS. Two traits uncommon in WBS were observed, unilateral renal hypoplasia in the mother and a hemivertebra at L5 in the son.

Clinical diagnosis of partial duplication 7q.

We report on two sibs with partial dup (7q), a retarded 9-month-old boy and an aborted fetus of 17 weeks' gestational age. Besides minor anomalies, the boy had frontal bossing, macrocephaly with hydrocephaly, a high forehead, and a large fontanelle. GTG banded chromosomes showed a 14p+ abnormality. Because his mother carries a balanced, de novo translocation with a breakpoint in band 7q33, the boy has a duplication of the distal portion of band 7q33 and the segment 7q34----qter. Our findings suggest that the phenotype in terminal duplications of 7q may, in some patients, be recognized clinically.

Girl with phenotypic abnormalities and a de novo, apparently balanced translocation 46,XX,t(5;10)(q35.2q11.2).

We describe a three-year-old girl with a triangular face, epicanthus, midfacial hypoplasia, apparently low-set ears, a small mouth with thin vermilion border, and a small chin, hypermobile joints, developmental delay with insecure gait, dystonic movement disorder, speech defect, and a history of unexplained undernutrition. She has a de novo, apparently balanced translocation t(5;10)(q35.2;q11.2). Using fluorescence in situ hybridization (FISH), we located the breakpoints in the 1.5-Mb area defined by YAC 753f5 (5q35.2) and within the approximately 2-Mb interval between 10cen and YAC 933a3 (10q11.21).

Boy with celiac disease, malformations, and ring chromosome 13 with deletion 13q32-->qter.

We describe a 2(1/2)-year-old boy with a ring chromosome 13 with distal deletion of 13q32-->qter and celiac disease.